Assay for trichothecenes

ABSTRACT

An immunoassay for trichothecenes that have at least three hydroxyl groups at specified positions is disclosed. It relies on developing antibodies to close trichothecene variants that are missing at least one of the hydroxyl groups, and then using these antibodies to test specimens in which the trichothecene has been converted to the variant (usually to the OAC variant). For example, DON and T-2 tetraol can be assayed for using this invention.

This invention was made with United States Government support awarded bythe Department of Defense, Grant Number DAMD-82-2021. The United StatesGovernment may have certain rights in this invention.

This invention relates to an assay for testing for the presence ofcertain trichothecenes. The invention involves the use of immunoassaytechniques in testing for trichothecenes that have at least threehydroxyl groups in specified ring positions.

BACKGROUND OF THE INVENTION

Naturally occurring trichothecenes are a group of toxic secondary fungalmetabolites which are produced by a number of fungi in the generalFusarium, Trichoderma, Myrothecium and Stachybotrys. Some natural andsome man made examples of trichothecenes are described in FIGS. 1 and 2.As shown in FIG. 1 of the attached drawings, trichothecenes contain acommon skeleton with different side chain residues at points R₁ -R₅.Three typical classes of natural trichothecenes are

type "A" (where R₅ is H or ISV), type "B" where R₅ is ═O, and type "C"where R₂ is linked to R₃ by a carbon chain.

Because of their toxic effects and the frequent contamination of thesetoxins in foods and feeds, trichothecenes are potentially hazardous tohuman and animal health. There is also concern regarding the possibleuse of these compounds as biological warfare compounds (e.g. in "yellowrain"). A dependable, sensitive, specific, simple, and inexpensivemethod for the detection of mycotoxins in foods, feeds, and biologicalfluids is therefore highly desirable. Testing for trichothecenes has inthe past been extremely difficult because this group of mycotoxins doesnot possess a chromophore group. Methods such as thin-layerchromatography, gas liquid chromatography, and mass spectrometry, whichhave been used for analysis of this group of mycotoxins are lacking insensitivity and/or specificity, or need expensive instruments. Also,extensive cleanup is generally necessary before actual analysis.

To overcome some of the difficulties encountered with these prior assaymethods, attempts have been made to develop immunoassays fortrichothecenes by utilizing the principle of specific antigen-antibodyinteraction. However, trichothecenes are small molecular weight organiccompounds which are too small to lead to desired levels of antibodydevelopment. Thus, they must first be conjugated to a protein or apolypeptide carrier in order for antibodies to be developed to them.Since natural trichothecenes do not have a reactive group that can bedirectly conjugated to protein, it is necessary to first introduce areactive group before the coupling reaction takes place. Approacheswhich have been used for conjugation involve the introduction of variousreactive groups at R₁ and R₅.

Such studies have led to the development of specific antibodies againstT-2, DAS, and DOVE. See FIG. 2 and F. Chu et al., 37 Appl. Environ.Microbiol. 104-108 (1979); G. Zhang et al., 51 Appl. Environ. Microbiol.132-137 (1986); R. Wei et al., 49 J. Fd. Prot. 267-271 (1986); F. Chu etal., 48 Appl. Environ. Microbiol. 777-80, 781-84 (1984). The disclosureof these articles and all other articles recited herein are incorporatedherein by reference as if fully set forth herein.

This approach has problems for trichothecenes such as DON, T-2 tetraol,and nivalenol. This is due to the presence of many hydroxyl groups atthe R₁ -R₅ positions. Such groups not only create problems inconjugation to proteins, but also appear to render the molecule lessimmunogenic (or may produce antibodies that are currently undetectable).

Thus, it can be seen that a need exists for an improved immunoassay fortrichothecenes which are of the type where at least three of the R₁ -R₅moieties are OH groups.

Summary Of The Invention

In one aspect, the invention provides an assay for the presence in asample of a trichothecene having at least three hydroxyl groups at theR₁ -R₅ positions. The assay comprises the steps of removing at least oneof the hydroxyl groups from the trichothecene (e.g. conversion toanother group) to form a variant which has no greater than two hydroxylgroups at the R₁ -R₅ positions, and then using an antibody capable ofbinding to the variant to test for the presence of the variant in themodified sample.

If one has first tested the original sample using that antibody (todetermine a control level in the sample before the removal of the OHgroup), the test of the modified sample will provide a measure of thepresence of multi-hydroxyl trichothecene. In the alternative, theantibody can act as a screen for the presence cf any one of manytrichothecenes (including the multi-hydroxyl one).

The preferred manner of removing the OH group(s) is to expose thetrichothecene to acidic anhydride in the presence of pyridine oraminopyridine, which will convert the OH group(s) at the R₁ -R₅ positionto acetate groups. The preferred trichothecenes are DON (which has threehydroxyl groups), T-2 tetraol (which has four hydroxyl groups), andnivalenol (which has four hydroxyl groups). It is expected that theassay will also work with other hydroxylated trichothecenes that arecurrently not amenable to immunoassay techniques.

The nomenclature R₁, R₂, R₃, R₄, and R₅ is defined as shown in FIG. 1.

In another form, the invention comprises a kit with an antibody capableof binding to Tri--Ac--DON, and a radioactively labelled compound (e.g.tritiated Tri--Ac--DON) that the antibody also competitively binds to.Another kit can be provided in which the antibody is capable of bindingto T-2 TA.

The manner of obtaining the antibodies is discussed in detail in thepreferred embodiment section of this application. In short, the variant(e.g. in which the OH groups have been converted to acetate groups) isaltered to create a reactive site and then conjugated to a protein suchas BSA. The conjugate is then used to immunize a rabbit (or otheranimal) so as to produce the antibodies of interest. Using what have nowbecome conventional techniques, monoclonal forms of the antibody canthen be obtained.

An object of the invention therefore includes providing an immunoassayof the above kind in which trichothecenes having at least three hydroxylgroups can be assayed for.

Another object is to provide an assay of the above kind which is simple,relatively inexpensive, and easy to perform.

Another object is to provide kits for conducting assays of the abovekind. Still other objects and advantages of the present invention willbe apparent from the description which follows.

DESCRIPTION OF THE DRAWINGS

A better understanding of the present invention will be accomplished byreference to the drawings. It should be understood, however, that thedrawings and the description of the preferred embodiments are merelyexamples of the invention. They are not intended to represent the fullscope of the invention. Rather, the claims should be looked to in orderto determine the full scope of the invention.

FIG. 1 depicts the basic chemical structure of trichothecenes, with theletters R₁, R₂, R₃, R₄, and R₅ designating substitution positions on thebasic framework;

FIG. 2 is a chart describing some of the more common trichothecenes;

FIG. 3 is a schematic depiction of the chemistry involved in thedevelopment of the antibodies for the DON assay; and

FIG. 4 is a schematic view of the chemistry involved in the developmentof antibodies for the T-2 tetraol assay.

In the drawings, abbreviations are as follows: TA is tetra-acetate, OAcis OCOCH₃, ISV is OCOCH₂ CH(CH₃)₂ and CMO is ═NOCH₂ COOH.

Description Of The Preferred Embodiments Example 1 P DON Assay

Deoxynivalenol (DON) is one of the trichothecenes which causes feedrefusal and other problems in swine. It is sometimes called "vomitoxin".FIG. 2 shows that DON has OH's at the R₁, R₃, and R₄ positions, and a ═Oat R₅.

As shown in schematic form in FIG. 3, the approach involved for DON isto first obtain antibodies against Tri--Ac--DON. One does this byobtaining Tri--Ac--DON and then converting Tri--Ac--DON to7,8-dihydroxycalonectrin (DHC) (see FIG. 2), a compound having OH at theR₄ and R₅ positions. This compound is then converted with ahemisuccinate group substituting for the OH at R₅. The resultingcompound is then linked to bovine serum albumin (BSA) and injected intorabbits to form the antibody. A description in somewhat more detail ofthis technique is described in our article G. Zhang et al., 49 J. Fd.Prot. 336-339 (1986) (not prior art).

Materials And Methods

DON was produced by Fusarium Graminearum strain NRRL 5883 in crackedcorn at 25° C. using the conditions described by R. Vesonder et al., 16Process Biochem. 12-15 (1980). The toxin was extracted from the culturewith methanol and purified on a silica gel column as describedpreviously in G. Bennett et al., 58 J. Am. Oil Chem. Soc. 1002A-1005A(1981). Primary DON standard was provided by Northern Regional ResearchCenter, U.S. Department Of Agriculture, Peoria, Ill.

DON--triacetate (Tri--Ac--DON) was prepared according to the methoddescribed previously. K. Ehrlich, 48 Microbiol. 1053-1054 (1984). Bovineserum albumin ("BSA"; RIA grade) was purchased from Sigma Chemical Co.(St. Louis, Mo.). Tritiated DON was prepared in a manner analogous tothe method of making tritiated DOVE described in Chu et al, 48 Appl.Environ. Microbiol. 781-784 (1984). The basic technique is toselectively oxidize the R₃ hydroxyl group to ═O at -20° C. for 30minutes, and then to reduce the ═O group with tritiated sodiumborohydride at -20° C. for 30 minutes. Tritiated Tri--Ac--DON wasprepared by acetylation of tritiated DON with acetic anhydride in thepresence of pyridine. Water soluble carbodiimide,1-ethyl-3,3-dimethylamino-propyl-carbodiimide (EDPC), was obtained fromAldrich Chemical Co. (Milwaukee, Wis.). Complete Freund's adjuvantcontaining Mycobacterium tuberculosis (H37 Ra) and incomplete Freund'sadjuvant were obtained from Difco Laboratories (Detroit, Mich.). Albinofemale rabbits (2.27 kg) were purchased from Klubertanz Rabbit Farm(Edgerton, Wis.) and tested to be Pasteurella negative before use. Allchemicals and organic solvents were reagent grade or better.

Preparation Of 7,8-Dihydroxycalonectrin ("DHC") And Hemisuccinate ("HS")Of DHC

7,8-Dihydroxycalonectrin was prepared by reduction of Tri--Ac--DON withsodium borohydride using the conditions described previously in F. Chuet al., 48 Appl. Environ. Microbiol. 781-84 (1984), and was purified bypreparative TLC. Succinylation of DHC was achieved by reaction of DHCwith succinic anhydride in the presence of dimethylaminopyridine underthe conditions analogous to those described for diacetoxyscirpenol. F.Chu et al., 48 Appl. Environ. Microbiol. 777-780 (1984). The compoundwas isolated by preparative TLC.

Conjugation of DHC-HS to BSA was done in the presence of water solublecarbodiimide (EDPC) using procedures analogous to those describedpreviously for T-2 toxin in F. Chu et al., 37 Appl. Environ. Microbiol.104-108 (1979).

Production Of Antibody

Immunization schedule and methods of immunication were essentially thesame as those described for T-2 toxin in F. Chu et al., 37 Appl.Environ. Microbiol. 104-108 (1979) using the multiple injectiontechnique in three rabbits. The rabbits were each injected intradermallyat multiple sites on the back with 2.0 μg of immunogen (DHS-HS-BSA)which was prepared by emulsifying 500 to 600 μg of the immunogen in 0.5ml of sterilized phosphate-saline buffer (PBS, 0.1M sodium-phosphatebuffer containing 0.9% NaCl, pH 7.5) with 1.5 ml of complete adjuvant.

Preimmune serum was obtained from each rabbit before immunization.Bleedings and booster injections were made at appropriate times afterinitial injection. For booster injection, 250 to 500 μg of antigen in1.0 ml of PBS were emulsified with 1.0 ml incomplete adjuvant and wereinjected into the thigh. The collected antisera were precipitated with(NH₄)₁ 2SO₄ to a final saturation of 33.3%. The precipitates wereredissolved in water and reprecipitated two times. Finally, theprecipitates were reconstituted to one-half of the original volume with0.1M PBS, dialyzed against distilled water for 30 min., then against0.01M phosphate buffer (pH 7.0) overnight at 6° C. and lyophilized.

Radio immunoassay ("RIA")

Basically, our RIA procedure involves incubation of specific antibodysimultaneously with a solution of unknown sample or known standard, anda constant amount of a competing radioactively labelled toxin (usually atritiated variant of the compound the antibody binds to). Afterseparation (usually by precipitation) of the free from the bound toxin,the radioactivity in the respective fractions can then be determined.The toxin concentration of the unknown sample is determined by comparingthe results to a standard curve. Several known methods, including theammonium sulfate precipitation method, double antibody technique, asolid phase RIA method in which the immunoglobulin G (IgG) wasconjugated to CNBr-activated sepharose gel, a dextrancoated charcoalcolumn and albumin-coated charcoal, have been used for the separation offree and bound trichothecene toxins in RIA.

A preferred protocol for Tri--Ac--DON is analogous to that described forT-2 toxin, in which an ammonium precipitation method was used toseparate the free and bound toxin. F. Chu et al., 37 Appl. Environ.Microbiol. 104-108 (1979). In general, 50 μl of radioactive Tri--Ac--DON(10,000 to 15,000 dpm) (the "hot" competitive compound) was incubatedwith 0.15 ml of antibody solution of various dilutions in phosphatebuffer (0.1M, pH 7.2) at room temperature for 30 min., and then in acold room (6° C.) overnight. Separation of the bound and free ligand wasachieved by an ammo ammonium sulfate precipitation method as describedin the Chu article. Radioactivity was determined in a Beckman modelLS-5800 liquid scintillation spectrometer in 5 ml of Aquasol (NewEngland Nuclear Corp., Boston, Mass.) for aqueous solution. It might benoted that the resulting antibody recognized several othertrichothecenes besides just Tri--Ac--DON.

RIA Assay For Natural Samples

With the availability of antibody against DON-triacetate, a radioimmunoassay for DON in natural wheat was developed. DON is extractedfrom the wheat sample with acetonitrile-water (84+16), defatted withhexane, and then reacted with acetic anhydride in pyridine to formDON-triacetate. The reaction mixture is loaded onto a C-18 cartridge toremove excess reagents and impurities. Acetylated DON is eluted from thecartridge with 50% methanol in water, and then analyzed by radioimmunoassay utilizing antiserum against DON-triacetate and tritiatedDON-triacetate. The limit of detection of DON was about 20 ppb. Analysisof twelve naturally contaminated wheat, corn, and mixed feed samples forDON revealed that RIA results agreed well with thin layerchromatographic analyses. For greater detail on this see our article seeour article. Xu et al., 69 J. Assoc. Off. Anal. Chem. 967-969 (1986)(not prior art). The assay will also show positive if mono-acetyl DON orbiacetyl DON was in the original sample.

ELISA Assay

Two types of ELISA assays (namely direct and indirect) have been usedfor analysis of some trichothecenes. See generally F. Chu, MycotoxinsAnd Phycotoxins (1986); F. Chu, 47 J. Fd. Prot. 562-569 (1984). Althoughboth types are competitive assays, the direct type ELISA uses atoxinenzyme conjugate as a marker as compared to the indirect system, inwhich a protein-toxin conjugate and a secondary antibody to which theenzyme has been conjugated, are used.

Direct Competitive ELISA

Horseradiah peroxidase (HRP) can be the enzyme conjugated withmycotoxins in the direct system. The antibodies are first coated to asolid phase, most commonly to the wells of a micrometer plate. Thesample solution or standard toxin is then incubated with enzyme-toxinconjugate in the antibody-coated microplate wells. After appropriatewashings, the amount of enzyme remaining bound to the plate is thendetermined by incubation with a substrate solution containing hydrogenperoxide and appropriate oxidizable chromogens. The resulting color isthen measured spectrophotometrically or by visual comparison with thestandards. Because of the competition of binding between free toxin andthe enzyme-toxin conjugate with the antibody in the microplate well, thepresence of toxin in the sample results in a decrease in enzymeconcentration, and subsequently yields less color. PG,13

Indirect Competitive ELISA

In the indirect ELISA, instead of using the toxin-enzyme conjugate,which may be prone to enzyme stability problems, a toxin-protein (orpolypeptide) conjugate is first prepared and then coated to themicroplate before assay. The plate is then incubated with specificrabbit antibody in the presence or absence of the homologous toxin. Theamount of antibody bound to the plate coated with toxin-proteinconjugate is then determined by reaction with goat anti-rabbitIgG-enzyme complex, which is generally commercially available, and bysubsequent reaction with the substrate. Thus, the protein-toxin coatedto the plate competes with the free toxin in their binding with theanti-toxin antibody, and the secondary anti-IgG-enzyme conjugate is usedas the marker. Both HRP and alkaline phosphates conjugated to the goatanti-rabbit-IgG have been used.

EXAMPLE 2

Our techniques for converting T-2 tetraol to T-2 tetra acetate, and thenforming antibodies to the resultant product are described in Wei et al.,160 Anal. Biochem. 399-408 (1987) (not prior art). The overall approachis schematically depicted in FIG. 4.

Synthesis Of T-2 Tetraol Tetraacetate

The first step is to convert all T-2 tetraol OH groups in the R₁ -R₅positions to OAC. A mixture of 66 mg of T-22 tetraol, 3 ml of drypyridine, and 3 ml of acetic anhydride was placed in a bottle with astopper at room temperature for 2 h. Five milliliters of ethanol wereadded to the reaction mixture and the solution was evaporated in vacuoto an oily product which was then dissolved in 3 ml of ethyl acetate andchromatographed on a column (1×19 cm) packed with 7 g of silica gel G-60(mesh 70-230, Merck AG, Darmstadt, FRG) and n-hexane.

The column was washed with 50 ml of n-hexane and then eluted with 100 mlof n-hexane-ethyl acetate (50:50, vol/vol). The eluate from 15th to 80thml which showed a single spot in thin-layer chromatography was pooledand concentrated in vacuo to a glassy solid that simultaneouslycrystallized. The colorless crystals weighed 88 mg, mp 177°-178° C.

Enzymatic Hydrolysis Of T-2 Tetraol Tetraacetate

To 3-Acetylneosolaniol (3-AC-NEOS)

The next step is to change the R₅ OAC to OH to provide a linker site.T-2 tetraol tetra-acetate was selectively hydrolyzed to 3-Ac-NEOS withpig liver S-9 according to the method of Yoshizawa et al., 46 Agric.Biol. Chem. 2613-2615(1982), except that the S-9 was prepared in a 0.25M sodium phosphate buffer (NaPB), pH 7.4, instead of Tris-HCl buffer. Ina 500-ml Erlenmeyer flask, 88 mg of T-2 tetraol tetraacetate crystalswere moistened with 1 ml of dimethylsulfoxide followed by addition of 6ml of ethanol and shaking at 37° C. to dissolve the crystals. Afteradding 120 ml of pig liver S-9, the mixture was incubated with shakingat 37° C. for 1 h. and the reaction was terminated by directly applyingthe reaction mixture to an Amberlite XAD-2 column (1.9×19 cm).

After washing with 300 ml of distilled water, the metabolites wereeluted from the column with 150 ml of acetone. The acetone eluate wasrotary evaporated to a few milliliters. Distilled water (10 ml) wasadded to the residue, which was then extracted with four 20-ml portionsof CHCl₃. The combined CHCl₃ extract was concentrated in a rotaryevaporator to an oily product, which was subsequently redissolved in asmall amount of CHCl₃ and chromatographed on a column (1×14 cm) packedwith 6 g of Florisil and equilibrated with CHCl₃. Stepwise elution with30 ml of CHCl₃ and 25 ml each of 1, 2, and 3% MeOH in CHCL₃ wasperformed. The eluate with 2% MeOH in CHCl₃ gave a single spot inthin-layer chromatography with a R_(f) value of 0.44 inchloroform:acetone (3:2, vol/vol) compared with the starting T-2 tetraoltetra-acetate, which had had a R_(f) of 0.63. This fraction weighed 57mg after it was evaporated to dryness.

Preparation Of Hemisuccinate Of 3--Ac--NEOS

The OH at R₅ is then used to add a linker. A hemisuccinate of3--Ac--NEOS was prepared by procedures analogous to those previouslydescribed for the preparation of T--2--HS in Chu et al., 37 Appl.Environ. Micro. 104-108 (1979). Briefly, 20 mg of 3--Ac--NEOS and 10 mgof 4-dimethylaminopyridine were dissolved in 1.3 ml of drytetrahydrofuran. Succinic anhydride (30 mg each) was added to thereaction mixture in the beginning of the experiment and then every 30min during a reflux period of 3 h. Thin-layer chromatography revealedthat almost all of the 3--Ac--NEOS was converted to a new product. Thereaction mixture was evaporated to dryness under nitrogen, redissolvedin CHCl₃, and washed with 0.1 N HCl and then distilled water. Thecombined CHCl₃ extracts were rotary evaporated to dryness, redissolvedin a small amount of CHCl₃ and chromatographed on a column (1×12 cm)packed with 5.3 g of Florisil and equilibrated with n-hexane. The columnwas stepwise eluted with 20 ml each of n-hexane, n-hexane:ethyl acetate(3:1, vol/vol), n-hexane: ethyl acetate:glacial acetic acid (40:60:1,vol/vol/vol) and ethyl acetate:glacial acetic acid (99:1, vol/vol). Theeluate with the last two solvents gave a single spot in thin-layerchromatography with a R_(f) value of 0.59 in ethyl acetate:MeOH:glacialacetic acid (88:10:2, vol/vol/vol). The product was confirmed to be3--Ac--NEOS--HS by mass spectral analysis.

The HS group is then linked to bovine serum albumin. Conjugation of3--Ac--NEOS--HS to BSA was carried out in the presence of a watersoluble carbodiimide (EDPC) using procedures analogous to thosepreviously described for T-2 toxin in Chu et al., 37 Appl. Environ.Microbiol. 104-108 (1979).

Production Of Antibody

Antibody to the conjugate was then produced. The immunization scheduleand methods of immunization were analogous to those described for T-2toxin Chu et al., 37 Appl. Environ. Microbiol. 104-108 (1979). Fiverabbits were used. Each rabbit was injected intradermally with 500 μg ofthe immunogen (3--Ac--NEOS--HS--BSA) in 1.0 ml of 0.1 M sodium phosphatebuffer (pH 7.4) containing 0.85% NaCl (PBS), with 2.0 ml of completeFreund's adjuvant. For booster injections, 500 μg of antigen in 1.0 mlof PBS and 2.0 ml incomplete Freund's adjuvant was used. The collectedantisera were precipitated with (NH₄)₂ SO₄ to a final saturation of33.3%. The precipitates were redissolved in water and reprecipitatedtwice. Finally, the precipitates were reconstituted to half of theoriginal volume with distilled water, dialyzed against distilled waterfor 0.5 to 1 h, then against 0.01 M PBS overnight at 6° C., and thenlyophilized.

Radioimmunoassay

Protocols for the assay were essentially the same as those described forT-2 toxin in which an ammonium sulfate precipitation method was used toseparate free and bound toxin. 3H--T--2 (tritiated) tetraol tetraacetatewas used as the marker ligand. It is prepared from tritiated T-2 tetraolby acetylation. Tritiated T-2 tetraol was prepared by hydrolysis oftritiated T-2. See Wei et al., 45 Biochem. Biophys. Res. Comm. 396-401(1971). Tritiated T-2 was made as described in Wallace et al., 25 J. Ag.Fd. Chem. 836-838 (1977). 50 μl of radioactive marker ligand (10,000 to12,000 dpm) was incubated with 0.15 ml of antiserum solution of variousdilutions in PBS at room temperature for 30 min and then in a cold room(6° C.) for 1 h or longer. Separation of bound and free ligand wasachieved by the ammonium sulfate precipitation method described earlierChu et al., 37 Appl. Environ. Microbiol. 104-108 (1979).

Radioimmunoassay Of Toxin In Corn

An appropriate amount of pure T-2 tetraol in acetone was mixed withwhite corn meal (Quaker) to final concentrations of 10, 20, and 50 ppb.The samples were dried at room temperature overnight to remove theacetone, and then subjected to extraction and cleanup. For eachanalysis, 1 g of sample in a 16×100-mm screw-capped test tube was mixedwith 5 ml of acetone in a Vortex Genie Mixer for 5 min and thencentrifuged. After centrifugation, 3 ml of the supernatant wastransferred to a vial to which 12 ml of distilled water was added. The20% acetone extract (total 15 ml) was then passed through a Sep-Pak C-18reversed-phase cartridge which had been previously washed with 5 ml ofacetone and then 20 ml of distilled water. After washing with 10 ml of20% aqueous acetone (washing discarded), the toxin was eluted from thecartridge with 2.4 ml of 60% aqueous acetone. The eluates were dilutedat least 10 times with PBS and subjected to RIA. The assay will alsoshow positive if T-2, H-T2, T-2 triol, 3'OH-T-2, or DAS is in theoriginal sample.

EXAMPLE 3

Another part of our work is described in T. Fan et al., 53 Appl.Environ. Microbiol. 17-21 (1987) (not prior art) where we describe meansfor preparing an antibody to CMO--T--2--4Ac (CMO is where R₁ is thelinker ═N--O--CH₂ -CO₂ H). This antibody also recognizes T--2--4--Ac andagain permits one to assay for T-2 tetraol by converting it toT--2--4--Ac with acetic anhydride in pyridine.

EXAMPLE 4

Monoclonal Techniques

A monoclonal antibody assay (as opposed to the above describedpolyclonal assays) can be developed. For example, a monoclonal antibodyagainst T-2 toxin has been produced by fusion of P3-NS1/1-Aq4-1 myelomacells with spleen cells of a BALB/c mouse which had been immunized with3-acetyl-neosolaniol, a derivative of T-2 tetraol tetraacetate,conjugated to BSA. A radioimmunoassay, using tritiated T-2 toxin anddiacetoxyscirpenol (DAS) as marker ligands, was used in screening thehybrid cells. One stable clone which produced antibody bound with bothT-2 toxin and DAS was obtained after screening and subcloning. Similarselection techniques should permit identification and development ofother monoclonals.

As can be seen from these examples, the problem in the art is solved byremoving (e.g. converting) interfering OH groups, and developingantibodies for the variant trichothecenes so created. Some of theantibodies so created will have broad trichothecene specificity, thuspermitting one to at the same time test for the presence of othertrichothecenes as well.

Since OH to OAC conversions of trichothecenes are readily accomplishedwithout affecting other immuno important groups, it is the preferred"removal" technique. However, still other removal techniques may alsoprove successful (e.g. substituting benzyl groups). Further, removal ofall OH groups at R₁ -R₅ is not always required. It is expected that goodresults can be achieved when less than three OH remain at R₁ -R₅positions on the variant.

Moreover, while DON and T-2 tetraol are specifically discussed herein,it should be appreciated that other trichothecenes having at least threehydroxyl groups should be amenable to these techniques. Further, theterm "trichothecene" is used herein in its broadest sense (e.g. coveringnot only natural compounds, but also man-made variants).

We claim:
 1. An assay for the presence in a sample of a trichothecenehaving at least three hydroxyl groups at the R₁ -R₅ positions, thetrichothecene having the formula: ##STR1## the assay comprising thesteps of: converting a hydroxyl group of the trichothecene to an organicmoiety linked to the remainder of the trichothecene by oxygen, so as toform a variant with no more than two hydroxyl groups at the R₁ -R₅positions; andadding an antibody capable of binding to the variant j anddetermining the presence of said trichothecene in said sample as ameasure of said antibody binding to said variant.
 2. The assay of claim1, comprising the further step of:first testing the sample using saidantibody to determine a control level in the original sample.
 3. Theassay of claim 1, wherein an OH group is converted to an ester in thevariant.
 4. The assay of claim 1, wherein a OH group is converted to OACin the variant.
 5. The assay of claim 1, wherein the trichothecene isdeoxynivalenol.
 6. The assay of claim 1 , wherein the trichothecene isT-2 tetraol.
 7. The assay of claim 1, wherein the trichothecene isnivalenol.